Nitric oxide (NO), produced by the inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of inflammation associated with several pulmonary disorders including asthma, interstitial lung diseases, lung infection, and septic shock. The long term goal of this research is to understand the regulation of iNOS activity and devise novel methods to regulate it. Our recent data indicated that iNOS is primarily degraded by the ubiquitin-proteasome pathway. Further, we showed that cellular iNOS is regulated by sequestration into a perinuclear aggresome. In this proposal, we would like to study two complementary molecular mechanisms for iNOS degradation. Our preliminary data identify CHIP (carboxyl terminus of Hsp70- interacting protein) to play a major role in regulating iNOS degradation by the proteasome as well as for targeting iNOS to the aggresome. Our data further suggest that ADRM1 (adhesion regulating molecule 1) serves as a receptor for iNOS at the proteasome interface. For this grant period, we propose to test the following hypotheses: a) iNOS is targeted to the proteasome by cooperative interaction between the ubiquitin pathway and the chaperone system, coordinated by CHIP and that the latter is critical for diverting excess iNOS to the aggresome. b). iNOS is handled at the proteasome by a specific receptor that deubiquitinates iNOS and delivers it to the proteasome. To test these hypotheses we propose studies with the following specific aims: 1) Elucidate the mechanisms by which CHIP regulates iNOS proteasomal degradation. Our preliminary data suggest that CHIP interacts with and ubiquitinates iNOS. They further show that CHIP promotes iNOS degradation by the proteasome pathway. We will study the underlying mechanisms for CHIP regulation of iNOS degradation. Further, our preliminary data suggest that CHIP is expressed in primary airway epithelial cells. We will further examine CHIP regulation in normal primary cells as well as cells obtained from subjects with airway inflammation of asthma. 2) Determine the role of CHIP in targeting of iNOS to the aggresome. Our preliminary data suggest a role for CHIP in triaging iNOS between the proteasome and the aggresome pathways. We will examine the molecular mechanisms governing these interactions. 3) Characterize the regulation of iNOS by ADRM1, a novel proteasome associated protein. Our preliminary data suggest that ADRM1 plays a role in iNOS degradation. We will determine if ADRM1 is a specific receptor for iNOS at the proteasome interface. The modulatory effects of ADRM1 on iNOS will be elucidated. The rationale for the proposed studies is that once these mechanisms are understood, they would greatly increase our understanding of cellular handling of iNOS and how NO levels are regulated. Future therapeutic strategies can be designed to regulate these cellular responses in disease states. PUBLIC HEALTH RELEVANCE. Nitric oxide, produced by the inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of inflammation associated with several pulmonary disorders including asthma, interstitial lung diseases, lung infection, and septic shock. The long-term goal of this research is to understand the regulation of iNOS activity and devise novel methods to regulate it. The rationale for the proposed studies is that once these mechanisms are understood, future therapeutic strategies can be designed to regulate these cellular responses in disease states.